This invention relates to the measurement of calcium using ion specific electrodes.
In biological fluids calcium ordinarily exists both in the form of calcium ions (Ca.sup.++) , and calcium complexed with protein and, to a lesser extent, other complexing agents in the fluids. Ion selective calcium electrodes measure only ionized, and not complexed calcium, despite the fact that it would be desirable in some situations to measure, in addition to ionized calcium, "total" calcium, i.e., the sum of ionized and complexed calcium.
It is known that complexed calcium can be released by lowering the pH of the sample containing the complexed calcium. The most efficient releasing of calcium (over 90% calcium released) is observed when a sufficient amount of a strong acid is added to the liquid to lower the pH below 3.0. Such a drastic lowering of pH, however, is inconsistent with optimal operation of conventional calcium electrodes.
In general, the present invention features a releasing reagent for releasing, as ionized calcium, complexed calcium in a liquid sample for measurement of such ionized calcium, together with any ionized calcium originally present in the sample, using a calcium ion specific electrode. The releasing reagent includes formic acid and formate salt (e.g., the sodium salt), in concentrations sufficient to render the pH to the sample, when mixed with the reagent, in the range of 4.0 to 5.5, most preferably 4.5.
The formic acid and formate salt together cause the release of calcium in biological samples. For samples, e.g., spinal fluid, in which complexed calcium is bound not to protein but to other constituents of the liquid, the releasing reagent need not contain ingredients in addition to formic acid and formate salt. For other samples, e.g., serum, which do contain appreciable amounts of protein, it is desirable that the releasing reagent contain an additional component which inhibits precipitation of the protein in the sample. Preferably, this precipitation inhibiting component is the salt of a strong organic acid, e.g., a haloacetate salt such as a monochloroacetate or trifluoroacetate salt. Of these latter two, trifluoroacetate salt is most preferred because of its superior long-term stability.
In particular embodiments of the invention, the concentration of formic acid is betweeen 0.105 Molar and 0.195 Molar, and is preferably about 0.15 Molar; and the concentration of formate salt is between 0.21 Molar and 0.39 Molar, and is preferably about 0.30 Molar.
When the releasing reagent contains monochloroacetate salt, this component is preferably present in a concentration of between 0.385 Molar and 0.715 Molar, most preferably about 0.55 Molar. When trifluoroacetate is present, it is preferably at a concentration of between 0.225 Molar and 0.416 Molar, most preferably about 0.32 Molar.
In other particular embodiments the reagent is for use in an apparatus in which the reagent is added to the sample at the same time as a reference solution is added to the sample; in this instance the reagent includes the reference solution, which is preferably ammonium chloride, preferably present in a concentration of between 1.5 Molar and 3.0 Molar, most preferably 2.0 Molar.
The releasing reagent of the invention provides efficient (greater than 99%) release of complexed calcium while avoiding detrimental extreme acidic conditions. Furthermore, the reagent, including or not including a reference solution, minimizes liquid junction and interference effects.